Non-coding RNA of salmonella and identification and use thereof
a salmonella and non-coding technology, applied in the field of biotechnology, can solve the problems of host fall ill, more dangerous drug-resistant strains, and lack of effective therapeutic drugs
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Benefits of technology
Problems solved by technology
Method used
Image
Examples
example 1
Screening of milRNA in Bacterium Infected Cells
[0162]Isolation and streaking for Salmonella strains are performed on an LB plate using an inoculating loop, medium-sized colonies are picked and inoculated onto a LB liquid medium, cultured at 37° C. overnight, then centrifuged to remove the LB medium, and the strains are resuspended with RPMI-1640+10% FBS medium.
[0163]One day before the infection with bacteria, human intestinal epithelial cells HT-29 digested with trypsin are plated in a cell culture dish, and cultured at 37° C. in a 5% CO2 incubator.
[0164]Cells are infected at a ratio of MOI (5-10:1), washed with PBS to remove the bacteria that have not been infected and entered the cells 2 hours after infection, then cultured with gentamicin-containing RPMI-1640+10% FBS, washed with PBS three times after cultured overnight, and cells are lysed with 0.1% PBST (PBS+Triton X-100), and filtered with 0.22 μm filter to remove the bacteria. Through the above treatment, the bacteria that ha...
example 2
The Ability of milRNA Inhibitors to Inhibit the Bacteria Infecting Hosts 2.1 In vitro Test
[0178]In this test, for the identified Salmonella specific sal-1, the correlation between it and the target gene inducible nitric oxide synthase (iNOS) is studied.
[0179]A target site region capable of binding with milRNA is amplified by PCR, double-digested by Spe I+Hind III and inserted into a pMIR-REPORT luciferase vector (purchased from Shanghai Innovation Biotechnology Co., Ltd.) to construct a recombinant plasmid (iNOS). In addition, the milRNA binding site of the above constructed recombinant plasmid is mutated to construct a recombinant mutant plasmid (iNOS mut).
[0180]Furthermore, the precursor of milRNA is loaded to an expression vector with CMV as a promoter, to construct a vector (pre-sal-1) overexpressing mi1RNA.
[0181]The milRNA control plasmid or pre-sal-1 and iNOS or iNOS mut are co-transfected into HEK-293T cells, and the reporter gene is detected, with the β-gal plasmid as a refe...
example 3
Use of Salmonella milRNA
[0200]The milRNA of the present invention is used for (a) the preparation of a reagent, a detecting chip or a kit for detecting Salmonella infections; or (b) the preparation of a regulator for regulating the iNOS expression or activity.
[0201]The nucleic acid chip for detecting Salmonella infections comprises: a solid-phase carrier; and oligonucleotide probes orderly fixed on the said solid-phase carrier, and the said oligonucleotide probes specifically capture the milRNA of the present invention.
[0202]The nucleic acid chip for detecting Salmonella infections of the present invention can screen with a high-throughput milRNA probes that stably change in serum, and furthermore predict and diagnose diseases through the overall change of milRNA in serum. The inventor firstly determines milRNA with no less than one copy in serum by the sequencing or quantitative PCR method, then synthesize reverse complementary probes of these milRNAs, and then spot these probes on...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More